protein concentration

In bionet.molbio.proteins,
Simon Penson <spensonwanson at nature.berkeley.edu> wrote
:James.Fethiere at BRI.NRC.CA (James Fethiere) wrote:
::> What would be the correct way of getting protein concentration from the
:> extinction coefficient. I have been told to just read the absorbance at
:> 280, sum up the individual extinction coefficients of Tyr, Trp, and Cys,
:> and use the formula to get my concentration. This seems pretty reasonable
:> to me, but i was wondering how the accuracy of this method compares to the
:> standard protein assay.
::There is no such thing as an `accurate' protein assay. Each protein
:behaves too differently in each assay system you choose. So, depending on
:how much time you want to spend on it, you'll get a rough or not so rough
:idea. The most accurate protein estimation method is the Kjeldahl total N
:method, but nobody uses this for routine biochemistry.
The Bradford and Bio Rad assays depend on the dye interacting with
basic residues in the protein; less basic residues, lower assayed
concentration. I'm not aware of anyone doing a sequence-based correction
factor for this.
One method that is fairly accurate is to purify and weigh. Of course,
you'll need a fair amount and must have all the salts removed. If you
have this, then you can correlate an A280 to the concentration and get
your extinction coefficient.
As for calculated A280s, I recently made up an RNase A sample from
"pure" Sigma RNase A. Based on mass, my concentration was 4.05 mM. Based
on the extinction coefficients in Gill & von Hippel (1989) Analytical
Biochemistry vol 182 p319, the concentration was 4.31 mM.
Wayne Baker (baker at iastate.edu) Maybe a great magnet pulls
Biochemistry & Biophysics All souls towards truth
Iowa State University -- k. d. lang, "Constant Craving"